X chromosome inactivation is the chromosome-based silencing mechanism eutherian mammals use to equalize the expression of X-linked genes between males and females early in development. To achieve transcriptional silencing of genes on 1 X chromosome each cell of a female must determine the number of X chromosomes it contains, choose 1 X to inactivate and initiate, propagate and maintain chromosome wide silencing. An elusive component of the X inactivation pathway is the initial choice between the 2 X chromosomes. In the mouse, choice is under the control of the X-controlling element (Xce), a genetically defined locus within the X inactivation center. Xce heterozygotes exhibit skewed, nonrandom inactivation of the X chromosome in somatic tissues. The objective of this proposal is to study X chromosome choice in mice. We previously conducted a phenotype-driven genetic screen involving chemical mutagenesis in the mouse and identified 3 genetically distinct autosomal mutations with dominant effects on X chromosome choice early in embryogenesis. This proposal will investigate the mechanism of X chromosome choice by: (1) Refining the map locations and cloning the genes responsible for the X chromosome choice mutations obtained in the genetic screen; (2) Isolating and characterizing embryonic stem cell lines from each of the mutant strains so that the earliest steps in the X inactivation process can be molecularly and biochemically monitored and manipulated; (3) Fine mapping and further characterizing the Xce. Elucidating the cis-acting sequences and the trans-acting factors involved in X chromosome choice will allow a greater understanding of the complex process of X inactivation. Furthermore, a better understanding of X chromosome choice will improve our knowledge of how differences in allelic ratios can result in significant phenotypic variation, including how unfavorable X chromosome skewing can lead to more severe phenotypes in a variety of X linked diseases.